Measurement assurance tools and potential application to alternative

test methods

Elijah Petersen and John ElliottCell Systems Science Group

Material Measurement LaboratoryNational Institute of Standards and Technology

NIST Practices• Measurements

• Develop new measurement methods• Improve accuracy/precision of measurements

• Reference Materials• Well-defined materials for use as a reference when

making measurements• Enables inter-lab comparability• Physical artifacts for calibrating instruments Food-matrix reference materials to

facilitate nutritional labeling

• Standards• Documentary standards, ASTM, ISO• Reference data (chemical spectra)• Technical Notes: “Guidelines for Evaluating and

Expressing the Uncertainty of NIST Measurement Results” (GUM)

NIST Synthetic RNA controls (ERCCs) used in sequencing of

Ebola virus genomes to characterize patterns of viral

transmission

• Biology/biotechnology• Cell-related measurements and technology (~1990)• Cytotox measurements, organism measurements

(~2005)2

Interlaboratory comparison with MTS assay

• 5 national metrology institutes were involved in the interlaboratory comparison

• Experimental design:• Share two A549 cell lines from ATCC and EMPA• Serum from local provider• Reagents from local provider• Serum and serum-free tests• Multiple replicates• Share nanoparticles (+ve PS) and chemical control

(CdCl2)

Elliott et al., Altex, 2017, 34(2), 201-218.

International standard (final draft international standard): ISO/DIS 19007: Nanotechnologies –In vitro MTS assay for measuring the cytotoxic effect of nanoparticles

NP EC50 values

-Looks like harmonization between the laboratories-No cell line differences-The serum conditions increases variability

CdSO4 EC50 values

Serum free conditions, variability less than with NPDifferences between cell lines

Misidentified Cell Lines Remain a Problem

Science. 2015 Feb; 347(6225): 938-40.

Assays Developed at NIST

STR genotyping• African green monkey (BMC Biotechnol. 2011 Nov 7;11:102.)

• Mouse – US Patent (Cytotechnology. 2014 Jan;66(1):133-47.)

• Chinese hamster (in progress)• Rat (in progress)

Multiplex PCR

targeting mouse STR

markers

NIH-3T3 STR Profile

Mouse Cell Line Authentication Consortiumhttps://www.federalregister.gov/documents/2016/10/13/2016-24768/notice-of-nists-mouse-cell-line-authentication-consortium

Consortium Members:National RepositoriesAcademic Institutions (National and International)Cancer Centers (National and International)Commercial Service Providers (National and International)

Consortium Goals:

1. Perform preliminary testing to determine if 19 mouse STR loci are useful

2. Conduct an interlaboratory study to collect STR genotyping data for 50 of the most commonly used mouse cell lines

3. Deposit STR profiles for mouse cell lines into the NCBI BioSample database

4. Publish a written consensus standard for mouse cell line authentication

1. Establish linear range in fluorescence scale using beads with assigned “equivalent number of reference fluorophore value (ERF)

Benc

hmar

k sc

ale

T

Benc

hmar

k sc

ale

urpose:

General Benchmarking of a Fluorescence Scale in Flow Cytometry

Cytometry Part A 73A: 279-288, 2008;Flow Cytometry Protocols: Third Edition, p53-65, 2011Current Protocols in Cytometry, 75:1.29.1-14, 2016; Flow Cytometry Protocols: Fourth Edition (in press)

wo step process:

2. Anchor the fluorescence scale (FS) to a benchmark material with the sample fluorophore

PStep 1: Provides evidence of linear range/proportionality on fluorescence scale

Step 2: Links relative intensity scale to a single reference material, provides reasonable instrument independent transferable scale.

Challenges in ERF assignments• Interlaboratory comparisons between bead manufacturer’s indicate

this measurement is challengingIssues in background subtraction, spectral correction of fluorometer, light scattering, bead counting and characterization, low fluorescence signals

500-fold difference!

• NIST has now developed a ERF assignment service

Flow Cytometry Quantitation Consortium81 Federal Register 136 (15 July 2016), pp. 46054-46055

ERF Value Assignment to Cytometer Calibration Microspheres Submitted by Consortium Members

How to assign ERF values to beads?

Light obscuration and flow cytometry methodsfor accurate microsphere concentration, fraction of fluorescence signal in single spheres

Different manufacturers’relative intensity beads

NIST SRM 1934FluoresceinNile redAllophycocyanin (APC)Coumarin 30

Calib

rate

d flu

orom

eter

Calib

rate

d flu

orom

eter

Emission of RF

Emission of bead

ERF/bead

ERF= Equivalent number of reference fluorophore

Conclusions

• Measurement assurance tools add robustness to a measurement process

• Interlaboratory comparisons, sensitivity analysis and process controls are valuable

• Robustness of Cell line ID, photoactivity testing and MTS viability assays have been validated with interlaboratory comparison

• Measurement assurance tools for flow cytometry including bead calibration underway

CollaboratorsPhotoactivity assay interlaboratory comparisonVytas ReipaVince HackleyBlaza TomanNam Woong SongPaul WesterhoffHaruhisa KatoKanokwan NontapotJonghoon Choi

Flow cytometer bead calibrationLili WangPaul DeRoseAdolfas Gaigalas

MTS assay interlaboratory comparisonMatthias RossleinHarald KrugPeter WickCordula HirstRawiwan ManiratanochoteNam Woong SongFrancois RossiAgnieska Kinsner-OvaskainenBlaza TomanJohn ElliottElijah Petersen

Cell line IDJamie AlmediaKen Cole